Raúl E. Russo

Modulation of plateau properties in dorsal horn neurones in a slice preparation of the turtle spinal cord.

1. Modulation of plateau properties in dorsal horn neurones was studied in a transverse slice preparation of the spinal cord of the turtle. In plateau‐generating neurones high frequency stimulation of the ipsilateral dorsal root (10‐20 Hz, 0.5‐2 min) produced a slow depolarization (2.9 +/‐ 0.6 mV, mean +/‐ S.E.M.; n = 6) and enhanced the properties mediated by dihydropyridine‐sensitive Ca2+ channels. The tetanic stimulus facilitated wind‐up and after‐discharges even when fast synaptic transmission was blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10‐20 microM), (+/‐)‐2‐amino‐5‐phosphonopentanoic acid (AP5, 100 microM), bicuculline (10‐20 microM) and strychnine (5‐20 microM). 2. Application of cis‐(+/‐)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (ACPD, 10‐50 microM) produced a slow depolarization (5.9 +/‐ 0.5 mV, n = 21) accompanied by an increase in input resistance (28.8 +/‐ 5.1%, n = 12). 3. ACPD increased the excitability by facilitating the plateau properties. In the presence of tetrodotoxin (TTX, 1 microM) a lower threshold and a slower decay of the plateau potential were observed. These effects resulted in facilitation of wind‐up and prolonged after‐discharges. 4. All ACPD‐induced effects were blocked by alpha‐methyl‐4‐carboxyphenylglycine (MCPG, 0.5‐1 mM), a selective antagonist of metabotropic glutamate receptors. The selective agonist for the type I metabotropic glutamate receptor ((RS)‐3,5‐dihydrophenylglycine (DHPG, 50 microM)) reproduced all the effects of ACPD. 5. Application of a supposed neuromodulator, substance P (1‐2 microM) produced a transient depolarization (4 +/‐ 0.6 mV) lasting 4‐6 min during continued application of substance P. Variable effects on the input resistance were observed, a slight increase (12 +/‐ 2%) being the most frequent. In 61% of the cells, substance P induced a clear increase in excitability with no detectable change in input resistance or membrane potential. 6. The effects of substance P on plateau properties were indistinguishable from those produced by ACPD. Unlike the transient depolarization, the facilitation of the plateau properties persisted in the presence of the agonist. 7. The substance P‐induced facilitation of the plateau potential was blocked by GR 82334 (5‐10 microM), a selective NK‐1 tachykinin‐receptor antagonist, and was not affected by MEN 10376 (2 microM), a selective NK‐2 antagonist. 8. The facilitation of plateau properties produced by dorsal root stimulation was also reduced by antagonists of metabotropic glutamate receptors and NK‐1 tachykinin receptors. 9. We propose that modulation of postsynaptic plateau properties in dorsal horn neurones by activation of type I metabotropic glutamate receptors and NK‐1 tachykinin receptors is involved in processing nociceptive information.

Enigmatic Central Canal Contacting Cells: Immature Neurons in “Standby Mode”?

The region that surrounds the central canal of the spinal cord derives from the neural tube and retains a substantial degree of plasticity. In turtles, this region is a neurogenic niche where newborn neurons coexist with precursors, a fact that may be related with the endogenous repair capabilities of low vertebrates. Immunohistochemical evidence suggests that the ependyma of the mammalian spinal cord may contain cells with similar properties, but their actual nature remains unsolved. Here, we combined immunohistochemistry for cell-specific markers with patch-clamp recordings to test the hypothesis that the ependyma of neonatal rats contains immature neurons similar to those in low vertebrates. We found that a subclass of cells expressed HuC/D neuronal proteins, doublecortin, and PSA-NCAM (polysialylated neural cell adhesion molecule) but did not express NeuN (anti-neuronal nuclei). These immature neurons displayed electrophysiological properties ranging from slow Ca2+-mediated responses to fast repetitive Na+ spikes, suggesting different stages of maturation. These cells originated in the embryo, because we found colocalization of neuronal markers with 5-bromo-2′-deoxyuridine when injected during embryonic day 7–17 but not in postnatal day 0–5. Our findings represent the first evidence that the ependyma of the rat spinal cord contains cells with molecular and functional features similar to immature neurons in adult neurogenic niches. The fact that these cells retain the expression of molecules that participate in migration and neuronal differentiation raises the possibility that the ependyma of the rat spinal cord is a reservoir of immature neurons in “standby mode,” which under some circumstances (e.g., injury) may complete their maturation to integrate spinal circuits.

Dynamics of intrinsic electrophysiological properties in spinal cord neurones.

The spinal cord is engaged in a wide variety of functions including generation of motor acts, coding of sensory information and autonomic control. The intrinsic electrophysiological properties of spinal neurones represent a fundamental building block of the spinal circuits executing these tasks. The intrinsic response properties of spinal neurones--determined by the particular set and distribution of voltage sensitive channels and their dynamic non-linear interactions--show a high degree of functional specialisation as reflected by the differences of intrinsic response patterns in different cell types. Specialised, cell specific electrophysiological phenotypes gradually differentiate during development and are continuously adjusted in the adult animal by metabotropic synaptic interactions and activity-dependent plasticity to meet a broad range of functional demands.

Inhibitory control of plateau properties in dorsal horn neurones in the turtle spinal cord in vitro

1 The role of inhibition in control of plateau‐generating neurones in the dorsal horn was studied in an in vitro preparation of the spinal cord of the turtle. Ionotropic and metabotropic inhibition was found to condition the expression of plateau potentials. 2 Blockade of γ‐aminobutyric acid (GABAA) and glycine receptors by their selective antagonists bicuculline (10‐50 μM) and strychnine (5‐20 μM) enhanced the excitatory response to stimulation of the dorsal root and facilitated the expression of plateau potentials. 3 Bicuculline and strychnine also facilitated the generation of plateau potentials in response to depolarizing current pulses, suggesting the presence of tonic ionotropic inhibitory mechanisms in turtle spinal cord slices. 4 Activation of GABAB receptors also inhibited plateau‐generating neurones. The selective agonist baclofen (5‐50 μM) inhibited wind‐up of the response to repeated depolarizations induced synaptically or by intracellular current pulses. 5 Baclofen reduced afferent synaptic input. This effect was not affected by bicuculline or strychnine and was blocked by the selective GABAB receptor antagonist 2‐hydroxysaclofen (2‐OH‐saclofen, 100‐400 μM). 6 Postsynaptically, baclofen inhibited plateau properties. Activation of GABAB receptors produced a hyperpolarization (7.0 ± 0.5 mV, mean ± s.e.m., n= 29) with an associated decrease in input resistance (22.7 ± 3.1 %, n= 24). These effects were blocked by extracellular Ba2+ (1‐2 mM). 7 When the baclofen‐induced hyperpolarization and shunt were compensated for by adjusting the bias current and the strength of the stimulus, baclofen still inhibited generation of plateau potentials. Wind‐up and after‐discharges were also inhibited by baclofen. These effects remained in the presence of tetrodotoxin (1 μM) and were antagonized by 2‐OH‐saclofen. 8 The inhibition of plateau properties was observed even when the baclofen‐induced hyperpolarization and shunt were blocked by Ba2+ and when potassium channels were blocked by Ba2+ (3 mM), tetraethylammonium (TEA, 15 mM) and apamin (0.25‐0.5 μM). The baclofen‐sensitive component of the plateau potential was reduced by nifedipine (10 μM), suggesting a modulation of postsynaptic L‐type Ca2+ channels. 9 We suggest that inhibitory regulation of plateau properties plays a role in somatosensory processing in the dorsal horn. The inhibitory control of wind‐up and after‐discharges may be particularly significant in physiological and therapeutic control of central sensitization to pain.

Connexin 43 Delimits Functional Domains of Neurogenic Precursors in the Spinal Cord

The cells lining the central canal (CC) of the spinal cord derive from the ventral part of the neural tube and, in some vertebrates, are responsible for the functional recovery after spinal cord injury. The region that surrounds the CC in the turtle contains proliferating cells that seem to generate both glia and neurons. Understanding the biology of spinal progenitors with the potential to generate new neurons “in situ” is important for cell replacement therapies. Here, we aimed to identify and characterize precursor cells in the spinal cord of Trachemys dorbignyi. To evaluate the population of proliferating cells, 5-bromo-2′-deoxyuridine (BrdU) was injected every 4 h (50 μg/g, i.p.) during 24 h. We found BrdU+ nuclei around the CC with a higher density in the lateral quadrants, in which whole-cell patch-clamp recordings showed extensive dye coupling of cells. Carbenoxolone (100 μm) increased the input resistance, suggesting strong gap junction coupling among precursors. The expression of brain lipid binding protein (a marker of a subtype of radial glia) and Pax6 matched the location of clusters, suggesting these cells belonged to a domain of neurogenic precursors. These domains were delimited by a high density of connexin 43 (Cx43) located on the endfeet of CC contacting cells. Our findings indicate that spinal precursors share basic properties with those in the embryo and neurogenic niches of the adult brain, and support a key role of functional clustering via Cx43 in spinal cord neurogenesis.

Functional and molecular clues reveal precursor-like cells and immature neurones in the turtle spinal cord

In lower vertebrates, some cells contacting the central canal (CC) retain the ability to proliferate, leading the reconstruction of the spinal cord after injury. A better understanding about the nature of these cells could contribute to the development of novel strategies for spinal cord repair. Here, by combining light and electron microscopy, immunocytochemistry and patch‐clamp recordings, we provide evidence supporting the presence of precursor‐like cells and immature neurones contacting the CC of juvenile turtles. A class of cells expressed the ependymal and glial cell marker S100 and displayed morphological and electrophysiological features of radial glia: relatively low input resistance, high resting potential, lack of active membrane properties and extensive dye‐coupling. A second class of S100 reactive cells were characterized by a higher input resistance and outward rectification. Finally, some CC‐contacting cells expressed HuC/D – a marker of immature neurones – and fired action potentials. The coexistence of cells with functional properties of precursor‐like cells and immature neurones suggests that the region surrounding the CC is a site of active neurogenesis. It remains to be demonstrated by lineage analysis whether, as in the embryonic cerebral cortex, radial glia are the progenitor cells in the turtle spinal cord.

Neural reconnection in the transected spinal cord of the freshwater turtle Trachemys dorbignyi

This paper provides the first evidence that freshwater turtles are able to reconnect their completely transected spinal cords, leading to some degree of recovery of the motor functions lost after injury. Videographic analysis showed that some turtles (5 of 11) surviving more than 20 days after injury were able to initiate stepping locomotion. However, the stepping movements were slower than those of normal animals, and swimming patterns were not restored. Even though just 45% of the injured turtles recovered their stepping patterns, all showed axonal sprouting beyond the lesion site. Immunocytochemical and electron microscope images revealed the occurrence of regrowing axons crossing the severed region. A major contingent of the axons reconnecting the cord originated from sensory neurons lying in dorsal ganglia adjacent to the lesion site. The axons bridging the damaged region traveled on a cellular scaffold consisting of brain lipid‐binding protein (BLBP)‐ and glial fibrillary acidic protein (GFAP)‐positive cells and processes. Serotonergic varicose nerve fibers and endings were found at early stages of the healing process at the epicenter of the lesion. Interestingly, the glial scar commonly found in the damaged central nervous system of mammals was absent. In contrast, GFAP‐ and BLBP‐positive processes were found running parallel to the main axis of the cord accompanying the crossing axons. J. Comp. Neurol. 515:197–214, 2009.

Spatial domains of progenitor-like cells and functional complexity of a stem cell niche in the neonatal rat spinal cord.

During spinal cord development, progenitors in the neural tube are arranged within spatial domains that generate specific cell types. The ependyma of the postnatal spinal cord seems to retain cells with properties of the primitive neural stem cells, some of which are able to react to injury with active proliferation. However, the functional complexity and organization of this stem cell niche in mammals remains poorly understood. Here, we combined immunohistochemistry for cell‐specific markers with patch‐clamp recordings to test the hypothesis that the ependyma of the neonatal rat spinal cord contains progenitor‐like cells functionally segregated within specific domains. Cells on the lateral aspects of the ependyma combined morphological and molecular traits of ependymocytes and radial glia (RG) expressing S100β and vimentin, displayed passive membrane properties and were electrically coupled via Cx43. Cells contacting the ventral and dorsal poles expressed the neural stem cell markers nestin and/or vimentin, had the typical morphology of RG, and appeared uncoupled displaying various combinations of K+ and Ca2+ voltage‐gated currents. Although progenitor‐like cells were mitotically active around the entire ependyma, the proliferative capacity seemed higher on lateral domains. Our findings represent the first evidence that the ependyma of the rat harbors progenitor‐like cells with heterogeneous electrophysiological phenotypes organized in spatial domains. The manipulation of specific functional properties in the heterogeneous population of progenitor‐like cells contacting the ependyma may in future help to regulate their behavior and lineage potential, providing the cell types required for the endogenous repair of the injured spinal cord. Stem Cells2012;30:2020–2031

Cytological organization of the central gelatinosa in the turtle spinal cord

This paper deals with the cytological organization of the central gelatinosa (CG) in the spinal cord of juvenile (2–12 months) turtles. We found two main cell classes in the CG: one with characteristics of immature neurons, the other identified as radial glia (RG). The cells surrounding the central canal formed radial conglomerates in such a way that the RG lamellae covered the immature neurons. We found three major subpopulations of RG that expressed S‐100, glial fibrillary acidic protein, or both proteins. Electron microscopic images showed gap junctions interconnecting RG. As with the mammalian neuroepithelial cells, most CG cells displayed intrinsic polarity expressed by structural and molecular differences between the most apical and basal cell compartments. The apical zone was characterized by the occurrence of a single cilium associated with a conspicuous centrosomal complex. We found a prominent expression of the PCM‐1 centrosomal protein concentrated close to the central canal lumen. In the particular case of RG, the peripheral end feet contacted the subpial basement membrane. We also found “transitional cell forms” difficult to classify by the usual imaging approaches. Functional clues obtained by patch‐clamp recordings of CG cells defined some of them as already committed to follow the neuronal lineage, whereas others had properties of less mature or migrating cells. The CG appeared as a richly innervated region receiving terminal branches from nerve plexuses expressing γ‐aminobutyric acid, serotonin, and glutamate. The results presented here support our previous studies indicating that the CG is an extended neurogenic niche along the spinal cord of turtles. J. Comp. Neurol. 502:291–308, 2007.

Intrinsic membrane properties of spinal dorsal horn neurones modulate nociceptive information processing in vivo

Non‐technical summary  The dorsal horn of the spinal cord is the first site in the central nervous system where painful sensory information is processed before transmission to the brain. In vitro recordings in spinal slices established that this processing relies on both plasticity of synaptic connections and intrinsic electrical properties of dorsal horn neurones (DHNs). DHNs may generate plateau potentials, which underlie intense discharges and long‐lasting after‐discharges in response to a brief stimulation, and represent a putative endogenous mechanism for amplification of painful sensory inputs. Using patch‐clamp recordings in the anaesthetized adult rat, we show that DHNs do generate plateau potentials in vivo, which shape their responses to natural sensory stimulation. Moreover, we give direct evidence for the involvement of these amplification properties in both short‐term (windup) and long‐term sensitisation associated with neuropathic pain, raising the possibility that plateau potentials could be putative therapeutic targets to control spinal component of neuropathic pain.